Abstract Protein misfolding and its pathogenic consequences have emerged as important factors in human disease. Diverse protein conformational diseases of the eye, including cataract, retinitis pigmentosa, glaucoma, macular degeneration, lattice and granular corneal dystrophies, etc., follow a common structural and pathological pathway. Prevention of protein aggregation is crucial for the preservation of biological function. Under development as frontline therapies to prevent protein misfolding and the ensuing cellular dysfunction and death are various molecular chaperones, including small molecules and protein and peptide chaperones that selectively bind to and stabilize the target proteins. Recently, a mini-chaperone (DFVIFLDVKHFSPEDLTVK), a peptide derived from the ?A-crystallin chaperone site and identified in our lab, was shown to stabilize various misfolded proteins and alleviate the associated pathology, suggesting that the mini-chaperone could be developed as a ?universal chaperone? against diseases involving protein aggregation, oxidative stress, apoptosis, and inflammation. We propose to improve the in vivo therapeutic efficacy of the peptide chaperone and uncover its potential as a universal chaperone for use in protein conformational diseases. We hypothesize that (1) the therapeutic efficacy of the chaperone peptide can be improved by use of D-amino acids (to increase half-life) during its synthesis and conjugation of a cell-penetrating sequence (for rapid uptake) and (2) the specificity of the peptide chaperone can be enhanced with the hydrolysis and release of the active chaperone peptide by the action of stress-specific proteases only in intended cells and tissues. To test our hypothesis, we will synthesize and characterize the cell-penetrating and in vivo activatable mini-chaperones (CPIAMCs) and determine its chaperone activity in primary lens epithelial cells, ARPE-19 cells, and N27 neural cells, to evaluate its potential as therapeutic peptide chaperone. The Specific Aims of this project are ? Aim 1 (a) Synthesis and characterization of CPIAMC peptide(s) that will encompass delineating the cellular uptake, toxicity and in vivo activation of CPIAMCs; and (b) investigation of the CPIAMCs efficacy to protect cells from oxidative stress. AIM 2: (a) Determine the effects of CPIAMCs on the secretion and accumulation of two disease-causing mutants, myocilin (Y437H) (implicated in glaucoma) in TM-3 cells and transforming growth factor beta-induced protein (R124H) (implicated in granular corneal dystrophy) in corneal fibroblasts and (b) evaluate the ability of CPIAMCs to suppress unfolded protein response and associated toxicity in cells expressing the myocilin Y437H or TGF? ?induced mutant protein R124H. The novel strategy for the design of molecular chaperones will lead to new tools for the development of therapeutic agents for protein aggregation diseases, ultimately improving the quality of life of persons suffering from such diseases and saving of tens of billions of dollars each year in healthcare costs for these diseases.